Melanogenesis, production of the biological polymer melanin, is a widespread phenomena in nature occurring in most phyla from fungi to mammals. The black, brown, buff and Tyndall-blue pigments found in feathers, hairs, eyes, insect cuticle, fruit and seeds are usually melanins. Melanins have been assigned a photoprotective role in the skin, their role in the eye and inner ear is unknown. Melanins are also found in the mammalian brain where they are referred to as neuromelanins. The biological function of neuromelanins is unknown.
The stepwise biosynthesis of melanins is depicted in FIG. 1. Tyrosinase (E. C. 1.14.18.1) catalyzes two types of reactions involved in the biosynthesis of melanin: the orthohydroxylation of monophenols to catechols, which is referred to as cresolase activity, and the dehydrogenation of catechols to o-quinones, designated as catecholase activity. Molecular oxygen is used for the hydroxylation reaction. For this reason, tyrosinase acting on a monophenol is referred to as a "mixed function oxidase". Hayaishi, in "Biological Oxidation" (Singer, ed.) p.581, Interscience Publishers, New York (1968). As used herein tyrosinase refers to all enzymes possessing the above described enzymatic activity. Tyrosinases are also sometimes referred to as polyphenol oxidases.
As can be seen from the biosynthetic pathway of melanin depicted in FIG. 1, tyrosinase is essential to the production of melanin. Therefore, the ability of an organism to express significant quantities of tyrosinase activity is essential to the organism's in vivo production of melanin.
Tyrosinase is not naturally found in all organisms. Rather, tyrosinase has been found to occur in a relatively limited number of prokaryotes, is absent in a variety of higher plants and is generally confined to specific cells of the skin in higher animals but may also occur in interior tissue, such as the substantia nigra, eye and inner ear. Given the limited number of organisms that produce melanin, one objective of the present invention is to provide a means for introducing tyrosinase activity into organisms that are otherwise unable to produce melanin.
Even in those organisms where tyrosinase activity occurs naturally, such activity is generally present at low levels. As a result, melanin is generally produced in small quantities by those organisms that possess tyrosinase activity. It is therefore a further objective of the present invention to provide a means for enhancing the level of tyrosinase activity in organisms in order to enhance the in vivo production of melanin.
Several prior art references teach how to genetically engineer an organism to possess tyrosinase activity. In U.S. application Ser. No. 7/857,602, filed Mar. 30, 1993, which is incorporated herein by reference, Applicants teach a method for producing melanin from transformed microorganisms wherein a sequence encoding for tyrosinase is introduced into the organism. Microorganisms that have be genetically engineered to enhance their abilities to produce tyrosinases include, but are not limited to species of Streptomyces, Escherichia, Bacillus, Streptococcus, Salmonella, Staphylococcus, and Vibrio. For example, cloned tyrosinase genes from Steptomyces sp. have been shown to produce melanin pigments in culture. J. Gen. Microbiol. 129:2703-2714 (1983); Gene 37:101-110 (1985). The cloned genes have also been expressed in Streptomyces and E. coli. della-Cioppa, Bio/Technology 8:634-638 (1990). In each case, both tyrosinase and ORF438 were required for melanin production.
U.S. Pat. No. 4,898,814 issued to Kwon discloses a cDNA clone of human tyrosinase and claims a method of making human tyrosinase by expressing the cDNA in E. coli.
Many forms of tyrosinase from bacteria, such as Streptomyces, require an activator protein. For example, the mel locus of S. antibioticus has been shown to contain two open reading frames (ORF's) that encode a putative ORF438 protein (M.sub.r =14,754) and tyrosinase (M.sub.r =30,612). ORF438 and tyrosinase are thought to be transcribed from the same promoter in S. antibioticus. Bernan, et al., Gene 37:101 (1985). Both genes are required for melanin production. Bernan, et al., Gene 37:101 (1985). Based on genetic evidence, ORF438 protein has been shown to function as a trans-activator of tyrosinase. Lee, et al., Gene 65:71 (1988). It has been suggested that the ORF438 protein is involved in tyrosinase secretion, or it may function as a metallothionein-like protein that delivers copper to tyrosinase, Bernan, et al., Gene 37:101 (1985); Lee, et al., Gene 65:71 (1988). The mel locus of S. glaucescens has a nearly identical ORF sequence upstream of tyrosinase that probably serves a similar function. Huber, et al. Biochemistry. 24:6038 (1985); Huber, et al., Nucleic Acids Res. 15:8106 (1987). The existence of an ORF438 protein, however, has never been confirmed in vivo.
The melanin operons of S. antibioticus and S. glaucescens have been isolated and sequenced, and both share sequence homology and similar gene arrangement. The polypeptide sequence encoded by ORF438 (146 amino acids) in S. antibioticus is structurally and functionally equivalent to URF402 (134 amino acids) from S. glaucesecens Huber, et al. Nucleic Acids Res. 15:8106 (1987). Disruption of the URF402 coding sequence abolishes the melanin phenotype similar to that already known for ORF438. Recent evidence suggest that the ORF438 protein (and by analogy the URF402 protein equivalent) functions as a molecular chaperone for tyrosinase. Chen, et al., J. Biol Chem. 268:18710 (1993).
Tyrosinase has also been isolated and employed to produce melanin in vitro. For example, in U.S. application Ser. No. 7/982,095, filed Nov. 25, 1992, which is incorporated herein by reference, Applicants teach the production of melanin in vitro using a tyrosinase which is excreted from the microorganism during fermentation. In vitro production of melanin is dependant on the production of significant quantities of tyrosinase activity. Tyrosinase activity may be lost during isolation of the secreted tyrosinase due to a disruption of the tyrosinase--activator protein complex. It is therefore an objective of the present invention to stabilize the tyrosinase--activator protein complex in order to reduce tyrosinase activity loss during isolation and purification.
Fusion proteins have been synthesized to overcome instability and proteolytic degradation of a polypeptide of interest. Many eucaryotic proteins have been produced in E. coli as fusion proteins with E. coli polypeptides such as Beta-galactosidase. Beta-galactosidase can be used to protect the foreign passenger protein from degradation in E. coli. Somatostatin, the first eurcaryotic protein to be produced in E. coli was produced by fusing a synthetic gene to the entire Beta-galactosidase coding sequence (Itakura, et al., Science 198:1056 (1977)) and somatostatin was subsequently isolated by chemical cleavage of the fusion protein. When two independently funtioning polypeptides are fused into a single polyprotein by way of a synthetic gene fusion, the resulting gene construct can be engineered for expression behind a single promoter, ribosome binding site, and initiation codon. A gene fusion constructed in such a way can be placed in a single location within a chromosome or episomal element for subsequent expression of the fusion protein. Such fusion genes are inherently more stable because both gene sequences are coordinately expressed at high levels from a single promoter. Undesirable effects such as differential down regulation or rearrangement and deletion of one of the genes can thus be avoided.
Unfortunately, however, most fusion protein constructs are not functional. Proteins fold into conformationally active states as dictated by their primary amino acid sequence. Additional polypeptide sequences at the C- or N- terminus can interfere with proper folding, thereby preventing the formation of a biologically active protein. Enzymes typically fold into well defined three-dimensional conformations to allow the formation of a catalytic pocket that excludes potential substrate molecules of abnormal size or conformation, but allows access of substrate molecules with the correct three-dimensional structure. Many enzymes will not function as fusion polypeptides because the sequence extensions interfere with proper folding, or interfere by sterically blocking the substrate's access to the catalytic site.
The present invention relates to Applicants' recognition that it might be possible to construct a biologically active fusion enzyme coupling tyrosinase with an activator protein. It has been shown that histidine residues #102 and #117 of the ORF438 activator protein are critical for copper binding and delivery to the active site of tyrosinase. Chen, et al., J. Biol. Chem. 268:18710 (1993). In order to form a biologically active fusion enzyme between tyrosinase and an activator protein, both functional domains of tyrosinase and the activator protein must be correctly folded. Further, the fusion enzyme must be able to assume a structural conformation enabling the activator protein to intermolecularly deliver copper to the active site of the tyrosinase to form a biologically active tyrosinase. Based on the existing prior art, it is unclear whether such a fusion enzyme would be biologically active.